As an animal feed supplement, phytase has proved very effective in improving the bioavailability of phytate phosphorus and other minerals as well (Gentile et al., J. Anim. Sci. 81:2751-2757 (2003); Lei et al., J. Nutr. 123:1117-1123 (1993)) and reducing phosphorus excretion (Han et al., J. Anim. Sci. 75:1017-1025 (1997)).
Thermostability is a highly desirable property for phytase to survive high temperature exposure during the feed-pelleting process (Mullaney, Adv. Appl. Microbiol. 47:157-199 (2000)).
Phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyze the hydrolysis of phytate into myo-inositol and inorganic phosphate in a stepwise manner, and are added to animal feeds to improve the absorption of phosphorus and to reduce phosphorus excretion. Among many phytases, Escherichia coli phytase has a great potential for industrial applications with the advantages of an acidic pH optimum, high specific activity for phytate, and resistance to pepsin digestion (Greiner, R. et al., Arch. Biochem. Biophys. 303:107-13 (1993); Lei, X. G., and C. H. Stahl, Appl. Microbiol. Biotechnol. 57:474-481 (2001); Rodriguez, E. et al., Arch. Biochem. Biophys. 365:262-267 (1999b); Rodriguez, E., et al., Arch. Biochem. Biophys. 382:105-112 (2000); Wyss, M., et al., Appl. Environ. Microbiol. 65:367-73 (1999)). The second E. coli phytase gene, appA2 has 95% sequence homology to appA gene (Rodriguez, E., et al., Biochem. Biophys. Res. Commun. 257:117-23 (1999a)). The 1.3 kb appA2 gene encodes a protein of 432 amino acids with 3 putative N-glycosylation sites and its product has a molecular mass of 46.3 kDa after deglycosylation (Rodriguez, E., et al., Biochem. Biophys. Res. Commun. 257:117-23 (1999a)). The crystal structure of E. coli phytase contains a conserved α/β-domain and a variable α-domain, which is very similar to the overall fold of rat prostatic acid phosphatase, Aspergillus niger PhyA phytase, and pH 2.5 acid phosphatase from A. niger, despite low sequence homology (Lim, D., et al., Nat. Struct. Biol. 7:108-1323 (2000)).
Investigations into the structural basis for the protein stability have illustrated general factors governing the stability of proteins (Kumar, S., et al., Protein Eng 13:179-91 (2000); Querol, E., et al., Protein Eng. 9:265-71 (1996); Querol, E., et al., Protein Eng. 9:265-71 (1996); Vieille, C., and G. J. Zeikus, Microbiol. Mol. Biol. Rev. 65:1-43 (2001); Vieille, C., and J. G. Zeikus, Trends Biotechnol. 14:183-190 (1996); Yip, K. S., et al., Structure 3:1147-58 (1995)). They include increase in hydrogen bonds and ionic interactions, reduction of conformational strain, improvement of the packing of the hydrophobic core and enhanced secondary structure propensity. Based on the proposed thermostabilizations and detailed structural information, a series of attempts to improve the thermostability of proteins have been made using rational design. Rodriguez et al. (2000) added potential glycosylation sites to improve the thermostability of E. coli AppA phytase by site-directed mutagenesis. As a semi-rational approach, the consensus concept was applied to phytase to improve thermostability and catalytic efficiency (Lehmann, M., et al., Protein Eng. 13:49-57 (2000a); Lehmann, M., et al., Protein Sci. 9:1866-72 (2000b)). Structure-based chimeric enzymes were developed as an alternative to directed evolution to improve the thermostability of A. terreus phytase (Jermutus, L., et al., J. Biotechnol. 85:15-24 (2001)). However, few attempts have been made to improve the thermostability of phytases by directed evolution.
Although a number of successful examples of rational approach have been reported (Georis, J., et al., Protein Sci. 9:466-75 (2000); Howell, E. E., et al., Biochemistry 29:8561-9 (1990); Kim, T., et al., Appl. Environ. Microbiol. 72:4397-4403 (2006); Minagawa, H., et al., Eur. J. Biochem. 270:3628-33 (2003); Perl, D., et al., Nat. Struct. Biol. 7:380-3 (2000); Williams, J. C., et al., Protein Eng. 12:243-50 (1999)), such structure-based rational approach requires not only detailed information on the structures but also the ability to predict the proper site of substitution concerning an optimal amino acid to be substituted (Kim, Y. W., et al., Appl. Environ. Microbiol. 69:4866-74 (2003)). Directed evolution has emerged as an effective alternative to rational design of enzyme to engineer enzymes (Kuchner, O., and F. H. Arnold, Trends Biotechnol. 15:523-30 (1997); Williams, G. J., and A. Berry, The Biochemist 25:13-15 (2003)). It involves generating a vast library of the gene of interest by random mutagenesis such as error-prone PCR or DNA shuffling, followed by screening mutants for desired properties. This approach has been particularly successful in improving the thermostability of proteins. In recent studies, the half-life of subtilisin S41 at 60° C. was increased by 1,200-fold and melting temperature of the mutant increased by 25° C. over the wild-type after eight successive rounds of error-prone PCR and in vitro recombination (Wintrode, P. L., et al., Biochim. Biophys. Acta 1549:1-8 (2001)). Cherry et al. (1999) improved the thermostability of a fungal peroxidase by 110-fold by combining mutations from error-prone PCR and in vivo shuffling with those from site-directed mutagenesis (Cherry, J. R., et al., Nat. Biotechnol. 17:379-84 (1999)). Giver et al. (1998) reported a thermostable esterase which increased the melting temperature by 14° C. by using error-prone PCR and in vitro recombination.
Increasing the thermostability of phytase is a great benefit because diets for swine and poultry are commonly pelleted at high temperature (60-80° C.). Although naturally thermostable enzymes may be produced by thermophilic organisms, such thermophilic enzymes usually do not function well at the physiological temperature of animals (Vieille, C., and J. G. Zeikus, Trends Biotechnol. 14:183-190 (1996)). Alternatively, heat-stable variants may be engineered by rational design and/or directed evolution (Giver, L., et al., Proc. Natl. Acad. Sci. USA 95:12809-13 (1998); Pedone, E., et al., Protein Eng. 14:255-60 (2001); Spiller, B., et al., Proc. Natl. Acad. Sci. USA 96:12305-10 (1999); Sriprapundh, D., et al., Protein Eng. 13:259-65 (2000)). Previous studies have investigated the relationship between protein structure and thermal stability (Beadle, B. M., and B. K. Shoichet, J. Mol. Biol. 321:285-96 (2002); Georis, J., et al., Protein Sci. 9:466-75 (2000); Kumar, S., et al., Protein Eng. 13:179-91 (2000); Querol, E., et al., Protein Eng. 9:265-71 (1996); Vieille, C., and J. G. Zeikus, Trends Biotechnol. 14:183-190 (1996)). The consensus concept was applied to a fungal phytase as a semi-rational approach (Lehmann, M., et al., Protein Eng. 13:49-57 (2000a)), and led to the development of the consensus phytase based on 13 fungal phytase sequences that showed an increase in melting temperature (Tm) by 15-22° C. while maintaining specific activity for phytate. Later, a new consensus phytase was engineered to improve the catalytic efficiency by replacing amino acid residues in the active site with the corresponding residues of Aspergillus niger PhyA phytase (Lehmann, M., et al., Protein Sci. 9:1866-72 (2000b)). The unfolding temperature of the new consensus phytase was decreased by 7.6° C. as an expense of the increased catalytic properties. Meanwhile, structure-based chimeric enzymes were designed to improve the thermostability of A. terreus phytase (Jermutus, L., et al., J. Biotechnol. 85:15-24 (2001)). Based on the crystal structure of A. niger phytase, one α-helix of A. terreus phytase was replaced by the corresponding stretch of A. niger phytase. This replacement improved thermostability without changing enzymatic activity.